Temperature-dependent internal content release from liposomes was examined using di-oleoylphosphatidylcholine (DOPC)/cholesterol liposomes with encapsulated Pluronic F127 molecules. The interaction of Pluronic F127 with the lipid bilayer at elevated temperature causes the release of encapsulated contents. Content release was measured using fluorescent markers of two different sizes: small, carboxyfluorescein (CF), and large, bovine serum albumin-conjugated fluorescein iso-thiocyanate (BSA-FITC). Release of CF was studied using fluorescence de-quenching, while that of BSA-FITC was studied using fluorescence emission quenching due to fluorescence resonance energy transfer (FRET). Temperature-controlled complete internal content release was achieved at a precise temperature by controlling the concentration of the encapsulated Pluronic. Increasing cholesterol % in the liposome composition resulted in a sharper transition with temperature in content release. The onset temperature of content release increased with decrease in Pluronic concentration. For the same Pluronic concentration, the onset temperature also depended on the size of the encapsulated marker and was higher for larger markers. We have established that onset of content release is determined by the critical micellar temperature (CMT) of the Pluronic. Temperature-sensitive liposomes, made stealth using di-stearoyl(polyethylene glycol 5000) phosphatidylethanolamine (DSPEG5000PE) in conjunction with Pluronic F127, had similar temperature sensitivity and efficiency in content release compared to the non-stealth liposomes.